Leica Kreatech FISH probes Mode D'emploi page 17

Kreatech FISH probes
Troubleshooting
Alternative protocol for cytology specimens:
1. Pre-warm 50 ml of 2 x SSC / 0.5% Igepal (LK-105B) in a Coplin jar to
37 °C in a water bath. Place prepared slides in the Coplin jar and
incubate for 15 minutes.
2. Dehydrate slides in 70%, 85%, and 100% ethanol, incubate for
1 min each at RT. Air-dry at RT.
Proceed with Probe preparation.
Alternative protocol for probe denaturation (separate probe and
sample denaturation):
1. Prewarm 70% formamide / 2 x SSC, pH 7.0 to 72 °C.
2. Denature slides in 70% formamide / 2 x SSC, pH 7.0, incubate at
72 °C (±1 °C) for 2 min.
3. Dehydrate slides in ice cold (-20 °C) 70%, 85%, and 100% ethanol,
incubate for 2 min each. Air-dry at RT.
4. Denature probe mix at 90 °C for 10 min and directly place on ice.
5. Briefly spin down probe vial, vortex probe vial and then spin down
probe vial again.
6. Apply probe to denatured slide, cover with glass coverslip, seal
with rubber cement.
Proceed with Hybridization.
Frequently asked questions
I have weak or no signals
• Re-hybridize making sure that the probe has been mixed correctly
and that it is at RT before use.
• Re-hybridize making sure that the stringency wash (Wash Buffer I)
is at the right temperature (72 °C (±1 °C)).
• Account for temperature drop when adding slides to pre-warmed
reagents.
• Use a minimum of 10 μl of probe per 22 x 22 mm coverslip.
• Check microscope filters and light source are correct and in full
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